The ontogenetic saga of a social brain

Angel Roberto BARCHUK, Gabriele David dos SANTOS, Ricardo DIAS CANESCHI,

Delcio Eustaquio de PAULA JUNIOR, Lívia Maria Rosatto MODA

Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal deAlfenas (UNIFAL-MG), Campus Sede, Rua Gabriel Monteiro Silva 700, Alfenas, Minas Gerais CEP 37130-000, Brazil

Received 4 April 2017 – Revised 20 July 2017 – Accepted 16 August 2017

Abstract – Queen and worker honeybees differ in a number of life-history traits, including the size of certain brainregions, such as the mushroom bodies (MBs), which are larger in workers. However, during the larval period, thedifferential feeding offered to queens promotes faster brain development. As a result, members of this caste havelarger brains than workers. This developmental process is accompanied by the higher expression of severalneurogenic genes. Nonetheless, a caste-specific shift in relative brain growth occurs during the next developmentalstage. The suggested molecular underpinnings of this phenomenon are variations in hormonal environments, whichmay mediate higher cell death rates in the queen’s brain than in the workers’. The brain development of this highlyeusocial bee is thus a paradoxical case that may represent an evolutionary by-product of the reproductive division oflabour in species with female size diphenism.

Apismellifera / honey bee / development / caste / phenotypic plasticity

1. INTRODUCTION

Adult honeybee queens are easily distin-guished from workers based on their externalmorphology. The queens are rotund creatures thatmove slowly, whereas workers are comparativelysmaller and exhibit constant, agile movements.This first impression might suggest that queenshave large organs, of which the ovaries are fairrepresentatives. In fact, queens do develop a largebody with a large reproductive system, which isthe anatomical representation of their primary taskwithin a colony, i.e. reproduction. Workers, on theother hand, develop a specific morphologicalcharacter set that allows them to perform the in-numerable tasks associated with maintaining thecolony, such as foraging, nursing the brood, nestcleaning, nestmate recognition, and guarding.

The morphogenetic fields governing caste-specific systems and organ development havebeen the focus of research in recent decades. Indevelopmental biology, morphogenetic fields arediscrete regions of developing organisms thatfunction as the major units of development(Levin 2012; Gilbert and Barresi 2016). Sincethe general fate of their cells is genetically deter-mined, changes in these fields bring about evolu-tionary changes. One morphogenetic field that hasattracted attention since the early work of Erber(1975) is that of the honeybee brain. Studies onthe ontogenetic development of the braindiphenism found in adult honeybees have re-vealed an interesting phenomenon. The differen-tial nutritional inputs received by developing lar-vae boost the development of the Bbipotential^brain found in embryos and the initial phases oflarval development (primarily L1-2) into that ofpresumptive queens thus resulting in a biggerbrain compared to that of presumptive workers(Moda et al. 2013). This picture, nevertheless,shifts during subsequent developmental stages

Corresponding author: A. Barchuk, barchuk@unifal-mg.edu.brManuscript editor: Klaus Hartfelder

Apidologie (2018) 49:32–48 Review article* The Author(s), 2017. This article is an open access publicationDOI: 10.1007/s13592-017-0540-4

before adult emergence. Specifically, morphoge-netic reorganisations occur so that the brain,which is larger in queen larvae, is relatively small-er than that of workers in the pharate-adult stage(Figure 1). Lastly, development assures that thebrains of newly emerged bees of both castes arealike in size and, as the queen is larger, adultworkers feature a proportionally larger brain thanadult queens. This developmental phenomenondoes not have a straightforward biological expla-nation, and we will discuss some of its morpho-logical and molecular underpinnings in the fol-lowing sections.

2. HONEYBEE DIPHENISM ANDCASTE DIFFERENTIATION

The genus Apis differs from the extremely richgroup of bees based on their evolutionary trait ofhighly eusocial organisation, a feature shared withthe stingless bees (Meliponini). These highly eu-social bees show caste morphology in addition to

cooperative brood care, reproductive division oflabour and overlap of generations (Wilson 1971).These complex traits have molecular underpin-nings that may have evolved from solitary-livingbee species. In this context and as anevolutionary-developmental hypothesis, the exis-tence of evolutionarily conserved molecular path-ways (Bgenetic toolkits^) has been suggested.Such pathways would play a role in controllingthe feeding behaviour and reproduction of bothsolitary and highly eusocial insects and thuswould underlie the evolution of such complextraits (Toth and Robinson 2007). Genomic evi-dence, however, supports that the only molecularcharacteristic that plays a role in the evolution ofsociality is the increasing complexity of geneexpression networks, rather than the existence ofa gene-set for sociality. The transition from soli-tary to social living seems to have required anincrease in gene regulatory system capabilities,including a higher number of genes regulated byDNA methylation and gains in transcriptionfactor-binding sites. Many of such changes con-stitute targets of regulatory factors that act tocontrol brain gene expression and neurogenesis(Kapheim et al. 2015). Interestingly, the nervoussystem size is a morphological trait that developsdifferentially between honeybee castes. Honeybeeworkers have double the relative antennal surfacearea compared to queens, have more chemorecep-tive plates per antenna, and exhibit a higher num-ber of facets of their compound eyes (Michener1974). Accordingly, workers have larger antennallobes and mushroom bodies (MBs), altogetherconstituting a sensory armamentarium for workersto cope with the broad range of duties they need toperform in a colony (Menzel 2014). Workers alsohave special, Btool-like^ organs that allow them toperform these duties, such as a pollen press and acorbicula, a long proboscis, large hypopharyngealand mandibular glands, wax glands, Nasanov’sgland, and a straight and barbed sting. On theother hand, queens feature the completemorphophysiological set of a reproductive ma-chine, with large ovaries and spermatheca anddeveloped secretory glands for scents that controlworkers’ behaviour (Snodgrass 1910; Michener1974; Pankiw et al. 1998, Hoover et al. 2003;see also in Miklos and Maleszka 2011).

Figure 1. The brain size shift between developing Apismellifera queens andworkers. The queen’s brain duringthe larval stage develops more rapidly than theworker’s. This pattern shifts during the next stage (pu-pal/pharate-adult), with the end result being that newlyemerged queens and workers feature equal-sized brains,although the brain/body ratio favours workers. L3: thirdlarval instar; L4: fourth larval instar; L5: fifth larvalinstar; Pw: white-eyed pupa, unpigmented cuticle;Pbm: brown-eyed pupa, intermediary pigmented cuti-cle; Pbd: brown-eyed pupa, dark pigmented cuticle; NE:newly emerged adult. Values on the y-axis are arbitraryunits only to demonstrate the relative developmentaltrend (modified from Moda et al. 2013; see alsoSection 6. Honeybee brain development during thepupal and pharate-adult stages).

The ontogenetic saga of a social brain 33

The first working model of the developmentalprocesses that lead to caste differentiation in hon-eybees was published after an effort to synthesiseDNA microarray hybridization results and datafrom the literature (Barchuk et al. 2007). Themodel states that the quality and amount of foodthat the bipotential female larvae receive deter-mine the developmental trajectories they follow,ultimately giving rise to morphologically distinctqueens and workers. Specifically, differentialfeeding would differentially activate the nutrient-sensing pathway Tor (Target of Rapamycin),which would then differentially induce the syn-thesis of juvenile hormone (JH) in the corporaallata . This signalling pathway and that of Insu-lin/ILPs, as well as the differential availability ofnutrients in the haemolymph (systemic level re-sponse), would lead to the differential develop-ment of specific organs and systems (morphoge-netic fields level response, i.e. brain, ovaries, legs,etc.). This developmental process would requirethe participation of molecular effectors (e.g. geneexpression cascades), some of which would beregulated epigenetically by differential gene meth-ylation. High signalling levels would allow theprospective queens’ body to grow and guaranteethe development of large ovaries, as well as re-press the development of worker specific organs(leg structures, etc.) (Barchuk et al. 2007). Thismodel has proven to be useful, and virtually, allinteractions have been supported experimentally(Patel et al. 2007; Kucharski et al. 2008; Maestroet al. 2009; Mutti et al. 2011). All, that is, exceptfor the confounding data on nervous system de-velopment that have been obtained from microar-ray experiments using whole body samples.

The observed split in the developmental trajec-tories was suggested to occur early in larval life(Cameron et al. 2013), very likely due to signifi-cant differences in the nutrient constituents ofworker and royal jellies (Leimar et al. 2012).Pioneering work had already suggested the influ-ence of early acting food components in castedifferentiation in the honeybee (Asencot andLensky 1976), and this view recently receivedsupport by the identification of small key mole-cules in honeybee larval food, namely,microRNAs (Guo et al. 2013; see also Ashbyet al. 2016). Small hydrophilic molecules (e.g.

microRNAs in royal jelly), which are also presentin the heads of nurse bees, were suggested to drivequeen development (queen determinator) manyyears ago (Rembold and Hanser 1964; Weaver1966; see also Rembold et al. 1974). Anothersuggested promoter of caste differentiation is amember of the major royal jelly proteins,royalactin, although the activities of this proteinhave not been comprehensively integrated into themodel outlined above (Kamakura 2011).Royalactin has been suggested to promote queendevelopment via the activity of the epidermalgrowth factor receptor. There is, however, a vig-orous debate regarding the role of this signallingpathway in honeybee caste development(Kucharski et al. 2015; see also Buttstedt et al.2016 and Kamakura 2016).

Studies focusing on the morphogenetic fieldsof honeybee castes have primarily considered thereproductive system, especially the developmentof the ovaries, which are very large in queens(approximately 200 ovarioles) compared to thoseof workers (approximately 12 ovarioles)(Snodgrass 1910; Michener 1974). This ovariandiphenism develops during the larval stage, inwhich queen ovaries respond to the larvaloverloading of royal jelly by undertaking markedovariole development under the molecular protec-tion of juvenile hormone. This hormone wasshown to prevent cell death in queen ovaries.Worker ovaries, on the other hand, in response toa different nutritional input and low levels of JH,undergo extensive cell death, leading to differen-tial ovary development between honeybee castes(Capella and Hartfelder 1998; Schmidt Capellaand Hartfelder 2002; Antonialli and Cruz-Landim 2006; Lago et al. 2016). The molecularunderpinnings of another morphogenetic field,that of the hind leg structures, have recently beenrevealed. Bomtorin et al. (2012) showed that thedevelopment of the Bshaved tibia^ used byworkers to carry pollen (pollen basket) dependsupon ultrabithorax expression during prepupaland early pupal phases. This finding was thenconfirmed by Medved et al. (2014) using geneexpression interference by RNAi.

The honeybee brain, a third morphogeneticfield, has also attracted a good deal of scientificattention. In the animal kingdom, eusociality is

34 A. R. Barchuk et al.

phylogenetically considered an unusual trait,since it is nearly confined to insects (ants, wasps,termites, and bees; Wilson 1971), in addition tooccur in some crustaceans (Emmet et al. 2000)and mammals [mole-rats (Burda et al. 2000) andhumans (Foster and Ratnieks 2005)]. Eusocialinsects are capable of executing complex, cooper-ative, and altruistic tasks in a colony environment.The capacity of honeybees to learn how to reachfood sources and communicate that knowledge toconspecifics makes them powerful models to un-derstand the basis of cognition (Chittka and Niven2009; Menzel 2012). Moreover, their small, plas-tic, and versatile brain allows researchers to tracethe routes of memory formation at cellular andmolecular levels (Menzel 2014; Cristino et al.2014; Haberkern and Jayaraman 2016). Thus, itis worth noting here some of the general aspects ofhoneybee brain biology. We will subsequentlypresent and discuss the morphological and molec-ular aspects that underlie the ontogenetic saga ofthe honeybee brain.

3. THE ADULT HONEYBEE BRAIN

The honeybee nervous system consists of alarge cephalic ganglion, the brain, which is con-nected to a series of segmental ganglia along theventral nerve chord (Snodgrass 1935). The honey-bee brain is a small organ consisting of 1 millionneurons with their neuropils, structures that arecomposed of axons, dendrites and glial cell pro-cesses that form a dense region of synapses. Theneuropils comprise three regions of the brain: theprotocerebrum, deutocerebrum, and tritocerebrum.The deutocerebrum essentially contains the anten-nal lobes, where 60,000 odorant receptor cells areconcentrated. The tritocerebrum consists of twobilateral small lobes that innervate the labrum andthe digestive tract. Lastly, the protocerebrum ischaracterised by two optic lobes, which are respon-sible for the conduction of visual image informa-tion to higher centres; the central body, a neuropilthat controls locomotor behaviour; and a pair ofMBs, which are critical elements responsible forlearning (Snodgrass 1935; Strauss and Heisenberg1993; Menzel 2012). The MBs are higher-orderprocessing centres that integrate stimuli from theoptic lobes and antennal lobes. The mushroom

bodies are composed of two calyces, each withthree concentric regions: the lip, where olfactoryinformation is received; the collar, which receivesvisual information; and the layered basal ring,where visual and olfactory inputs converge. Be-cause of their architecture, the calyces are alsoknown as Bcup-like^ neuropils, which contain hun-dreds of Kenyon cells (KCs). The KCs are dividedinto three subpopulations according to the inputsensory stimulus: visual, olfactory andmechanosensory. The KCs dendritic arborizationsspread to the calyces, and their axonal projectionsform the peduncles, which are divided into alpha(vertical) and beta (median) branches, also termedthe alpha and beta lobes (Mobbs 1982; Kenyon1896; Farris et al. 1999; Strausfeld 2002).

Because of their physiological plasticity, thehoneybee MBs have been extensively studiedthroughout the entire development of adult behav-iour in workers. There is a decrease in KC volumeand an increase in MB volume associated withforaging (Withers et al. 1993). No neurogenesishas been observed in adult brains, and studiesusing Golgi impregnation showed that intrinsicMB dendrites of older workers were typicallymore spread out and longer than those of youngbees. These characteristics apparently reflect theeffects of learning and memory in terms of den-dritic growth of intrinsic neurons and neuropilvolume (Farris et al. 2001; Fahrbach et al.1995a; Withers et al. 1993). In comparison, thearchitecture of a queen’s brain changes duringadulthood (from 1 day to 5 months old). Specifi-cally, the population of KCs decreases by approx-imately 30%, and the MB volume increases be-tween 25 and 50%. In this context, an importantfeature shared by both castes is exposure to highlevels of JH prior to neuropil development, sug-gesting that anatomical changes in KCs and MBsare under endocrine control (Robinson 1992;Fahrbach et al. 1995b).

4. EMBRYOLOGICAL ORIGIN OFTHE HONEYBEE BRAIN

In 1915, J.A. Nelson published a monograph of282 pages and 95 figures that represented anddescribed various aspects of honeybee develop-ment. Depending on temperature, development

The ontogenetic saga of a social brain 35

from oviposition to larval eclosion takes 70–76 h,and this period is divided into 15 stages based onmorphological characteristics (Nelson 1915).Some years later, Du Praw refined Nelson’s workusing innovative techniques and established 10stages of honeybee embryogenesis (Du Praw1967). Later, using scanning electron microscopy,Fleig and Sanger (1986) observed that honeybeenervous system development begins 44 h afterfertilisation, at Du Praw’s stage 9. Two longitudi-nal thickenings or ridges extending along the ven-tral side of the embryonic germ band go on toform the ventral nerve cord. Cephalically, a pro-liferative area in the head ectoderm gives rise tothree prominences, the protocerebrum,deutocerebrum and tritocerebrum (Nelson 1915;Du Praw 1967; Fleig and Sanger 1986). Theseswellings compose the brain neuropils, the archi-tecture of which is composed of neurons, theirprocesses, and glial cells.

Using grasshopper embryos as a model,Wheeler (1891) studied the histogenesis of neuro-genic tissues and identified the progenitors ofneurons and glia, which are termed neuroblasts(NBs). Wheeler observed that after gastrulation,the ventral ectoderm (or neuroectoderm), consistsof two kinds of cells, NBs and dermatoblasts,which will form the integumentary system. TheNBs start to bud off smaller cells, the ganglioncells, by mitotic division. Other researchers havereported the same observations in Orthoptera,Dermaptera, the mason bee and beetles (Wheeler1891; Nelson 1915). Since this time, experimentsperformed with Drosophila melanogaster haveelucidated many of the molecular and cellularmechanisms related to nervous system develop-ment, with a focus on NB specification, differen-tiation and segregation (Knust and Campos-Ortega 1989; Campos-Ortega and Hartenstein1997; Doe 1992; Urbach and Technau 2003).Signalling mechanisms that are governed by spe-cific families of genes that include pre-patterning,proneural, and neurogenic genes, orchestrate thedelamination of NBs from the neuroectoderm(Knust and Campos-Ortega 1989; Campos-Ortega and Hartenstein 1997; Hartenstein andWodarz 2013). There are two types of NBs, typeI and type II. Type I NBs divide asymmetricallyand give rise to both another NB—in a process

termed self-renewal—as well as a smaller cellreferred to as a ganglion mother cell (GMC).The GMC is committed to differentiate into aneuron and glial cell. Type II NBs can also self-renew or can form intermediate neural progenitors(INPs), which give rise to GMCs (Boone and Doe2008; Homem et al. 2015; Altenhein 2015;Koniszewski et al. 2016).

Some aspects of nervous system developmentare conserved in hemi- and holometabolous in-sects. The spatial arrangement, timing of delami-nation, and molecular network that specifies theNBs are strongly conserved between species(Urbach and Technau 2003; Koniszewski et al.2016). On the other hand, NBs that form specificneuropil structures, such as the MBs, differ be-tween species in number, arrangement, and pro-liferation behaviour. In A. mellifera , a compactgroup of 40–45 NBs per hemisphere forms theMBs, whereas 2–6 NBs are found in Diptera,Hemiptera, and Neuropteroidea (Malun 1998;Farris et al. 1999; Ito and Hotta 1992; Cayreet al. 1996). In the following section, we discusstheir participation in the development of the larvalhoneybee brain.

5. HONEYBEEBRAINDEVELOPMENTDURING THE LARVAL STAGE

The brain of a recently hatched honeybee larvais similar to that of other insects and does not seemto exhibit morphological differences between thecastes much before the fourth larval instar, L4(Nelson 1924; Moda et al. 2013). This lack ofobserved differences, however, might be due tomethodological limitations and the challenge in-volved in dissecting such small organs. During theearly larval development, the honeybee brain al-most completely fills the head capsule and con-sists of two symmetrical proliferating halves witha central mass of nerve fibres, all surrounded by aneurilemma (Nelson 1915, 1924; Farris et al.1999). The most prominent part of each hemi-sphere is the ontogenetic derivative of theprotocerebrum, with its conspicuous optic lobe.The halves of the protocerebrum are joined bynerve fibres, forming the supraoesophageal com-missure. The deutocerebrum and tritocerebrumderivatives seen during this period are very small

36 A. R. Barchuk et al.

and become organised below the protocerebrum,representing the precursors of the antennal lobesand, together with the suboesophageal commis-sure, form a ring that surrounds the oesophagus(Nelson 1924).

Comprehensive studies on postembryonicbrain development in A. mellifera were publishedby Malun (1998) and Farris et al. (1999). Theseauthors used basic histological analyses combinedwith BrdU incorporation and immunolocalizationof a MB neuropil marker (cAMP-dependentprotein kinase type II) and myocyte enhancerfactor 2, a marker of noncompact KCs. Usingthese techniques, the morphological developmen-tal dynamics of worker MBs was described fromthe first instar larva to adult eclosion. These stud-ies showed that the brain in L1 and L2 (seeTable I) features two clear NB clusters (prolifera-tion centres for KCs) per hemisphere, a smallmedial cluster with approximately 16 cells and alarge lateral cluster with approximately 45 cells(Malun 1998; Farris et al. 1999; see also Urbachand Technau 2003). Both clusters are delimitedfrom the rest of the brain mass by a thin layer ofearly GMCs (see also Hähnlein and Bicker 1997).There are no calyces, lobes, or pedunculi observedat these developmental phases. From the thirdlarval instar to metamorphosis, the NB clustersundergo a steady increase in both volume andthe number of cells, including some scatteredGMCs. These two types of proliferating cells areresponsible for giving rise to the organisation ofthe brain. A NB divides asymmetrically to giverise to another NB and a smaller GMC, the latterof which divides symmetrically into two KCs(Farris et al. 1999). Thus, at the third larval instar,it is possible to identify the first manifestation ofpeduncular neuropile ramifications, indicating thedifferentiation of the first KCs (future outer com-pact cells, originated by mitoses of GMCs migrat-ing from the NB cluster). This is clearly seen fromthe fourth larval stage onward, when these cellscome to surround the NB clusters and form a thinsheath of small cells that extend axons and formthe primordia of the branching pedunculi (Farriset al. 1999). All of these structures continue toincrease in size and thickness, and by the spinningphase of the fifth instar (L5S), a distinct layer ofGMCs becomes apparent between the NB cluster

and the surrounding sheath of KCs. In addition,the pedunculi that originate from the base of eachcluster during the preceding fifth instar feedingphase (L5F) become more evident (Farris et al.1999). The morphogenetic field of the MBs isthus formed by concentric layers of proliferativecells. Knowledge of the development of the MBsand other honeybee brain regions such as the opticlobes (Marco Antônio and Hartfelder 2016) hasbroad scientific interest given that, for example,pesticides used in agriculture to control insectstarget the brains of larval bees (Tomé et al. 2012;Tavares et al. 2015).

Table I. Developmental phases of A. mellifera consid-ered in this work (for details see Rembold et al. 1980and Michellete and Soares 1993)

Abbreviation Developmental phase

E Embryo

L1 First instar larva

L2 Second instar larva

L3 Third instar larva

L4 Fourth instar larva

L5F1 Fifth instar larva, feeding phase 1

L5F2 Fifth instar larva, feeding phase 2

L5F3 Fifth instar larva, feeding phase 3

L5S1 Fifth instar larva, cocoon-spinning phase 1

L5S2 Fifth instar larva, cocoon-spinning phase 2

L5S3 Fifth instar larva, cocoon-spinning phase 3

PP1 Fifth instar larva, prepupa 1

PP2 Fifth instar larva, prepupa 2

PP3 Fifth instar larva, prepupa 3

Pw White-eyed pupa, unpigmented cuticle

Pp Pink-eyed/pharate-adult transition,unpigmented cuticle

Pdp Dark pink-eyed pharate-adult,unpigmented cuticle

Pb Brown-eyed pharate-adult, unpigmentedcuticle

Pbl Brown-eyed pharate-adult, lightpigmented cuticle

Pbm Brown-eyed pharate-adult, intermediarypigmented cuticle

Pbd Brown-eyed pharate-adult, darkpigmented cuticle

NE Newly emerged adult

The ontogenetic saga of a social brain 37

Using phalloidin and DAPI staining of whole-mount brains, Moda et al. (2013) showed clearpeduncular organisation in worker larvae in theL5F phase. In contrast, this organisation was al-ready observed to the end of L3 in queen larvae(Figure 2a). Moreover, these authors reported thatMB calyces first appear at the beginning of theL5S phase in workers and slightly earlier inqueens (L5F). This neuropil differential morpho-genesis is also accompanied by a marked differ-ence in MB cell proliferation. Cell proliferationrates, as assessed by EdU staining, increasinglydiffer between castes from L4 until L5S, differ-ence that favours queen larvae (Moda et al. 2013).Higher levels of brain neuron proliferation inqueen larvae were previously demonstrated by

Roat and Cruz Landim (2008). These authors,by measuring conventional histological sections,showed that the brain of queen L5 larvae featuresan approximately 45% larger NB area than thebrain of worker larvae, and this difference wasseen to persist until the middle of the pharate-adult developmental period. During the prepupalstage in workers, the number of NBs per cluster ofthe brain peaks at approximately 500; a significantnumber of KCs are generated and located exter-nally to the cluster (non-compact cells), and thecalycal neuropil appears along with the almostcompletely grown α and β lobes (Malun 1998;Farris et al. 1999). Information on general aspectsof honeybee larval development and metamor-phosis can be obtained fromCruz-Landim (2009).

Figure 2. The developmental dynamics of the larval brain favour the queen honeybee caste. a Summary of thetiming of morphological differentiation in queen and worker larval MB structures. Continuous lines, queens.Discontinuous lines, workers. b –f General transcription profiles of key genes in the larval differential braindevelopment between honeybee castes. b failed axon connection , c short stop , d krüppel homologue −1 , egalactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase P, f anaphase promoting complex 4 . Modifiedfrom Moda et al. (2013).

38 A. R. Barchuk et al.

The morphological dynamics of differentialbrain development observed between queen andworker larvae may be conceived as a natural ex-periment and, accordingly, the fate of the respec-tive morphogenetic fields should be molecularlyestablished by the differential expression of spe-cific genes. The first genes suggested to play arole in differential neurogenesis between honey-bee castes were reported by Barchuk et al. (2007).Nonetheless, as these genes were identified usinggenome-wide analyses of whole-body RNA sam-ples, they may not reflect the actual biology of asmall organ like the brain, whose contribution ofdifferentially expressed genes is likely to be min-imal. With this limitation in mind, Moda et al.(2013) carried out genome-wide analyses onRNA samples taken from L4 brains of queensand workers, identifying 16 differentiallyexpressed genes. RT-qPCR assays were used topinpoint five genes whose expression may beresponsible for the differential brain developmentbetween castes during the larval phases, with allfive being more highly expressed in queens(Figure 2b–f): failed axon connection (fax ),k r ü p p e l h o m o l o g u e – 1 ( k r - h 1 ) ,galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase P (GlcAT-P ), ana-phase-promoting complex 4 (APC -4), and shortstop (shot ). The differential expression of thesegenes was confirmed by immunolocalization(Moda et al. 2013). Fax, GlcAT-P, and shot proteinsare known to participate in fasciculation processes,usually leading to the development of neuropils inthe brain and in the peripheral nervous system(Liebl et al. 2000; Pandey et al. 2011; Reuteret al. 2003; see also Farris and Sinakevitch 2003for data on dachshund gene regulatory function inbrain development). kr-h1 , on the other hand, is aJH immediate response gene that also acts to inte-grate the ecdysteroid pathway and is known tohave functions associated with neurogenesis (Shiet al. 2007; Jindra et al. 2013). APC, combinedwith the coactivators Cdh1 and Cdc20, has func-tions beyond its classical mitotic role. For example,APC regulates neurogenesis, glial differentiationand migration, neuronal morphogenesis, survivaland metabolism, synapse formation and plasticity,as well as learning and memory (Huang and Bonni2016). Other neurogenic genes have also been

studied in the context of compound eye develop-ment in honeybee workers and drones (MarcoAntônio and Hartfelder 2016).

Taken together, the available morphologicaland molecular evidence indicates that the differ-ential neurogenesis favouring the brain develop-ment of the honeybee queens is due to earlydifferences in nutritional signalling betweenqueen and worker larvae. These differences pro-mote the establishment of caste-specific hormonalenvironments, which in turn drive the expressionof different groups of effector genes (Ulrich andRembold 1983; Hartfelder and Engels 1998;Barchuk et al. 2007; Maestro et al. 2009; Muttiet al. 2011; Moda et al. 2013). These may beconsidered to be the molecular responsible forthe development of the initially larger brain seenin queen larvae. Hence, this diphenism can beinterpreted as representing a heterochronic effectof differential larval feeding regimes, which aftermorphological reorganisations leads to the devel-opment of distinct nervous systems proper ofadult honeybee female castes.

6. HONEYBEEBRAINDEVELOPMENTDUR I NG THE PU PAL ANDPHARATE-ADULT STAGES

Brain structures and the distinct lip and collarregions of the MBs continue developing and in-creasing in size during the pharate-adult period ofdevelopment, ultimately reaching final adult orga-nisation and size. On the other hand, the numberof NBs and the volume of the NB clusters startdecreasing from the prepupal/pupal stages, both inqueens and workers (Malun 1998; Farris et al.1999; Roat and Cruz Landim 2008, 2010a, b).KCs continue proliferating, and by the beginningof pharate-adult development (Pb-Pbl), a thirdtype of cells appears and forms the inner compactcell layer. At this time, the morphogenetic field ofthe MB is formed by clusters of a few NBsBcapped’ by active GMCs, all surrounded bylayers of various types of KCs. During the nextdevelopmental phases of the pharate-adult period,the morphogenetic field starts to be interiorlyremodelled by cell death events suffered by theremaining proliferative cells (Malun 1998; Farriset al. 1999; Roat and Cruz Landim 2008, 2010a,

The ontogenetic saga of a social brain 39

b). The cell death events within NB clusters startimmediately following an abrupt cessation of cellproliferation (Ganeshina et al. 2000). Using BrdUincorporation and TUNEL assays, these authorsshowed that both events are sequentially coupledand coincide temporally with a minor peak inecdysteroid titres during the middle of thepharate-adult period of development (Pinto et al.2002). The functional association betweenecdysteroids and the triggering of cell death inthe honeybee brain was further demonstrated bythe manipulation of hormone levels. Using 20-hydroxyecdysone injections in live pharate-adultbees and squash preparations of the brains, Malunet al. (2003) demonstrated that this hormone actsby reducing mitotic activity in NB clusters. Asimilar action of ecdysteroids on nervous systemreorganisation was demonstrated in the stinglessbeeMelipona quadrifasciata anthidioides, wherethese steroid hormones regulate cell death andventral nerve cord shortening (Pinto et al. 2003).

Interestingly, the observed differentialneurogenesis and rate of cell death in pharate-adult brains following the cessation of cell prolif-eration are caste specific. The MBs of queensexhibit far more cell death than those of workers(Roat and Cruz Landim 2008, 2010a), and differ-ential cell fates are also observed in other brainregions, such as the optic (Roat and Cruz Landim2010b) and antennal lobes (Roat and CruzLandim 2011). In all cases, these events favourthe development of larger structures in the workerbrain. This outcome has also been shown usingimmunostaining techniques with confocal fluores-cence microscopy. Groh and Rössler (2008) re-ported that the glomerular volume (and themicroglomeruli number in the MB-calyx lip) innewly emerged workers is slightly larger than inqueens, even though the development of the ol-factory synaptic neuropil of the antennal lobe andthe MB is approximately 4 days faster in queens.Interestingly, the number of microglomeruli in theMB-calyx is under brood temperature control,such that any variation from the normal intra-colony temperature promotes the development ofsmaller structures (Groh et al. 2004), therebypushing the resulting brain phenotype to that ofqueens. This finding led the authors to proposethat the observed accelerated development of the

queen olfactory pathway results in diminishedolfactory wiring in members of this caste, whichmight have physiological implications regardingolfactory learning skills in adult bees.

The observed beginning of caste-specific braindevelopment early in the pharate-adult stagemight be under combinatorial hormonal control.Ecdysteroid titres are higher in queens than inworkers during the entire larval stage and thepupal phase (Pw, see Rachinsky et al. 1990;Pinto et al. 2002) and seem to differ only slightlyin their profile during the subsequent pharate-adult stage. JH titres are also higher in queensduring the whole larval stage, reaching basallevels at the larval-pupal moult and remaining atthis level until the end of pharate-adult develop-ment (Hartfelder and Engels 1998). It is possiblethat neurogenesis is favoured in queen larvae by acombination of high nutritional input in the pres-ence of high levels of developmental hormones(ecdysteroids and JH), the production of which isthought to depend on nutrition. However, duringthe next developmental stage (larval-pupal moultand pupal-pharate-adult phases), the highecdysteroid titres that characterise queens, butnow in the absence of JH, push the cell death/proliferation ratio towards queens and now benefitworker brain development. The cell death-inducing effects of ecdysteroids and the protectiveeffects of JH have been demonstrated to play arole in other insect model systems, including opticlobe development in D. melanogaster (Riddifordet al. 2010; see Monsma and Booker 1996; Resset al. 2000;Wu et al. 2006; Parthasarathy and Palli2007, for examples in other model systems).

By applying comparative morphometric analy-ses of histological sections, we found that braindevelopment favouring workers begins as early asthe white-eyed, unpigmented cuticle pupa phase(Pw), immediately following the larval-pupalmoult (Figure 3a–b). This caste-specific mor-phometry continues through pharate-adult devel-opment, with brains showing a progressively larg-er total area in both castes. However, the differen-tial morphometry that favours pharate-adultworkers vanishes at the end of this developmentalperiod (Pbd-NE), when the brains of queens ap-pear to be slightly larger than those of workers(see Table I for developmental staging).

40 A. R. Barchuk et al.

MBs as a whole are pretty similar in size inboth castes at the Pw phase (actually, calices arestill a bit larger in queens, Figure 3e) and favourworkers during the following developmentalphases. This rule holds with the exception of theleft peduncular bodies, which experience a shift atPbm-Pbd, favouring queen brains (see Peduncular

Body in Figure 3d, f). Furthermore, right-sidecalices are quite similar in size in NE bees(Figure 3c). As there is no very clear size differ-ence in MBs regions between queens and workersin Pw, the observed difference in total brain areafavouring the worker in Pw is likely due to differ-ences in the antennal lobe and suboesophageal

Figure 3. The development of the pupal and pharate-adult brain favours the worker caste. In a and b , the total brainarea excludes that of the optic lobes (delimited by the outermost discontinuous line shown in b . Brains (n=5 perphase per caste) were dissected in 4% paraformaldehyde, dehydrated and embedded in Leica Historesin. The brainswere carefully sectioned (3 μm) in series, the largest slices (6–7, corresponding to the medium part of the brain)glued to a slide and the slice with the largest width of the brain was always used for measurements. The sections werestained with basic fuchsin and methylene blue, and the images were captured using a Zeiss Axio Lab microscopeequipped with Zeiss Axio Vision software. The areas were measured using ImageJ software (https://imagej.nih.gov/ij/). All measurements (different brain regions) were performed with the same bee samples. Pw, white-eyed,unpigmented cuticle pupa; Pdp, dark pink-eyed pharate-adult, unpigmented cuticle; Pbm, brown-eyed pharate-adult,intermediary pigmented cuticle; MBl and MBr, left and right MBs, respectively; PBl and PBr, left and rightpeduncular bodies, respectively; TA= total brain area. Means ± SE; two-way ANOVA followed by Bonferroni test,*p > 0.05; ***p > 0.001.

The ontogenetic saga of a social brain 41

ganglionic areas (Figure 3). A similar, thoughopposite explanation, can be applied to the varia-tion in total brain area favouring queens in Pbd-NE. This hypothesis is in agreement with theclassic view, which describes adult worker MBsas being larger than those of queens (seeMichener1974; Miklos and Maleszka 2011).

Morphometr ic data thus support theabovementioned findings regarding hormone actionon cell proliferation/death in pharate-adult brains.The clear pattern of brain size diminution after thePbm phase in both castes coincides with the previ-ous ecdysteroid peaks that are typical of this devel-opmental stage (Pinto et al. 2002). It is possible thata molecular cascade promoted by differential varia-tion in ecdysteroid levels leads to differential braincell death and survival between the castes, causingworker brains to become transiently larger thanthose of queens. Taken together, the morphometricdata support the classic notion of adult worker brainsbeing proportionally larger than those of queens(brain size/body size) and show that MBs per seare larger in workers than in queens (Figures 3 and4), with all of these differences appearing from thebeginning of the pharate-adult developmental stage(Pdp). AsMBs are the integrative centre of the brain,the observed difference in size that favours workersclearly fits with the demands of the broad behav-ioural repertoire of this caste. A similar morpho-behavioural correlation was found in other socialinsects, such as ants. In these insects, workers withthe broadest task repertoire and behavioural plastic-ity have the largest MBs, and those with the highest

degree of task specialisation have the smallest MBs(Feinerman and Traniello 2016). This trade-off hasevolutionary significance and aligns well with thebrain diphenism seen in honeybees.

The observed morphological differentiation inthe developing brain of queens and workers duringthe pharate-adult stage is currently under molecularinvestigation. Using a transcriptomic approach,Vleurinck et al. (2016) detected 1760 differentiallyexpressed genes (DEGs) in the brain tissue ofqueens and workers at Pdp phase. Of these genes,902 were upregulated in workers and 858 wereupregulated in queens. These DEGs were foundto be associated with several molecular processes,such as cell surface receptor signalling, RNA me-tabolism, DNA-templated transcription, phospho-rus metabolic processes, and nucleotide binding.Interestingly, almost all of the DEGs (1623) wereprotein-coding genes, with only 43 being found totranscribe non-coding RNAs. Of the total numberof DEGs, approximately 40% appear to be differ-entially spliced, with most of these being protein-coding genes (Vleurinck et al. 2016). Recently, ourgroup also used a genome-wide approach (micro-array hybridization) to identify more than 300DEGs in the brain of queens and workers at thebeginning of the pharate-adult stage (Pp), many ofthem related to neurogenic processes and cell deathprevention (unpublished). Additional studies arenecessary to pinpoint the extent to which eachthese genes participates in the differentialneurogenesis between pharate-adult honeybeecastes.

Figure 4. Mushroom body and peduncular body/brain ratios of Pw and pharate-adult queen and worker honeybees. aMB/brain ratios. b Peduncular body/brain ratios. For details on the histological analysis and description of thedevelopmental stages, see Figure 3. Means ± SE; two-way ANOVA followed by Bonferroni test, *p > 0.05;**p > 0.01; ***p > 0.001.

42 A. R. Barchuk et al.

7. CONCLUDING REMARKS

The saga of the female honeybee brain beginswith differential development that favours queensduring the larval stage, likely in response to dif-ferential nutritional input. This pattern shifts withthe larval-pupal moult. During the first part of thepharate-adult developmental period, workers de-velop larger brains than queens, possibly in re-sponse to differences in hormonal environmentsbetween the castes. Our working hypothesis isthat the observed differences in ecdysteroid levels,in the absence of JH during pupal and most ofpharate-adult development, are translated into dif-ferential gene expression profiles in the brain.These effects lead to di f fe rent ia l ce l lproliferation/death in the brains of the two castes.The hormonal environment would thus constitutethe physiological link between the nutritionallybased differential neurogenesis in the larval stageand the neurogenic processes taking place duringthe pupal/pharate-adult stage. Hormones are alsolikely the causal factors that transform a rapidlygrowing tissue during the larval stage into one thatexperiences far more cell death in the subsequentdevelopmental stage. This relatively long-lastingeffect of high nutritional availability in the pres-ence of high titres of ecdysteroids (but the absenceof JH) resembles a physiological process found inother systems. It has been shown that high levelsof TOR activity in the absence of growth factorspromote cell death by excess nutrients (Panieriet al. 2010). Similarly, nutrient excess may induceapoptosis through the action of high levels of theinsulin/insulin receptor pathway (Tachibana et al.2015). In the honeybee brain, the Bsurvival^ factorwould be JH, the titres of which remain at basallevels until the final phases of the pharate-adultstage (Rembold 1987; Hartfelder and Engels1998). Thus, it seems that a high nutritional inputduring larval development may subsequently re-sult in diminished neurogenesis, likely due tolower cell survival. This hypothesis is currentlybeing tested in our laboratory.

Another pending issue in the saga of brain de-velopment is that of the absolute MB size and thebrain volume/body volume ratio, which favournewly emerged workers. Does this phenomenonaccompany the evolution of sociality in bees?

There are data supporting the notion that size dif-ferences between female bees increase along withthe level of sociality. Hallmark examples, as pre-sented byMichener (1974), are the increasing trendin mean size variation among unfertilized workersand bees with enlarged ovaries in Augochloropsissparsilis (Augochlorini, a semisocial species). Thisstate is followed by the size dimorphism found inLasioglossum malachurum (Halictini, a primitive-ly eusocial species) and in Lasioglossum imitatum ,which features an additional sociobiological rele-vant characteristic. Specifically, female sizediphenism in this species is accompanied by aproportional allometry regarding ovary develop-ment (activated by oogenesis), including ovariolenumber (as in L. laevissimum , Packer 1992). With-in Allodapini, the Australian bee Exoneuravariabilis shows size differentiation betweenworkers and egg-layers, a difference that is alsoaccompanied by differential ovary activation. Inaddition, there are certain species of the genusBombus (also primitively eusocial) with remark-able differences in female size as well as in phys-iology, behaviour, and even coloration. The finalstage is represented by highly eusocial bees (e.g.the genus Apis , Apini, and the stingless bees,Meliponini; see Sakagami 1982). As in honeybees,there are differences in caste size that are reflectedin brain size differences, and there is a general trendin increasing caste size differences with increasinglevels of sociality. Brain development in the highlyeusocial honeybee might thus be a paradoxical casethat represents a by-product of the evolution ofreproductive division of labour in species withfemale size diphenism. Therefore, A. melliferacastes fit the social brain model, which states thatinteractions with conspecifics represent cognitivechallenges to animals and that any increase in socialcomplexity favours the development of largerbrains (Dunbar and Shultz 2007). Thus, it may bethat evolutionary caste differences in bees werepromoted by developmental morphophysiologicaltrade-offs in which the caste specialising in repro-duction invested in ovary development and func-tion, and the non-reproductive multi-task casteinvested in brain development (and the other be-havioural facilitating structures, such as the corbic-ula). To test if the evolution of this phenomenonoccurred in bees and thus agrees with the social

The ontogenetic saga of a social brain 43

brain hypothesis (wasps, for example, seem to fol-low an opposite pattern, O’Donnell et al. 2015),studies must be performed on the allometric rela-tionships between body regions in bees of differentsociality levels, as well as on the conservation ofthe gene expression networks that mediate honey-bee caste brain diphenism.

ACKNOWLEDGEMENTS

We thank Paulo EF Alvarenga (UNIFAL-MG),Marcela Laure, and Luiz Aguiar (FMRP-USP) for tech-nical assistance in sampling brains, as well as Zilá LPSimões, Marcia Bitondi and Erica Tanaka for helpfuldiscussions. We also thank Zilá LP Simões and KlausHartfelder for critically reading a previous version of theMS.

This research was supported by Fundação de Amparoà Pesquisa do Estado de Minas Gerais (FAPEMIG APQ-02134-14) and FINEP/PROINFRA 01/2008. ARB issupported by CNPq (Conselho Nacional deDesenvolvimento Científico e Tecnológico, Proc.307426/2014-9). We also thank Lisete M. Rosa (Sítiodas Rosas) and Prof. Antônio M. Siqueira (Fazenda daLagoa) for generously allowing the installation of ourexperimental apiary.

AUTHORS’ CONTRIBUTION

ARB conceived this research and designed exper-iments; LMRM participated in the design andinterpretation of the data; GDS, RDC and DEPJperformed experiments and analysis; ARB andLMRM wrote the paper. All authors read andapproved the final manuscript.

OPEN ACCESS

This article is distributed under the terms of theCreative Commons Attribution 4.0 International Li-cense (http://creativecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and repro-duction in any medium, provided you give appropriatecredit to the original author(s) and the source, provide alink to the Creative Commons license, and indicate ifchanges were made.

Ontogénèse d’un cerveau social

Apis mellifera / abeille / développement / caste /plasticité phénotypique

Die Ontogenese eines sozialen Gehirns

Apis mellifera / Honigbiene / Entwicklung / Kaste /phänotypische Plastizität

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